UHPLC Optimization Study for Improved LC-MS Performance and Throughput

Importance of the Topic

Ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) is essential for high-resolution, sensitive analysis in environmental, food safety and pharmaceutical applications. Optimizing UHPLC conditions enhances separation quality, reproducibility and sample throughput for automated workflows.

Objectives and Study Overview

This study compared three UHPLC configurations to assess their impact on retention time precision, peak characteristics and daily sample throughput. Eleven pesticide standards were analyzed using binary high-pressure gradient, quaternary low-pressure gradient and a 1500 bar high-pressure system with varying gradient lengths.

Methodology and Instrumentation

Stock solutions of 11 pesticides at 10 mg/mL were diluted 1:10000 for LC-MS analysis. Three Thermo Scientific Vanquish UHPLC systems were evaluated:

• Vanquish Horizon (1500 bar, binary high-pressure gradient)
• Vanquish Flex Binary (1000 bar, binary high-pressure gradient)
• Vanquish Flex Quaternary (1000 bar, low-pressure gradient, up to four solvents)

Columns used:

• Hypersil GOLD C18, 2.1×100 mm, 1.9 µm
• Accucore Vanquish C18+, 2.1×100 mm, 1.5 µm

Gradients ranged from 2 to 100 min at 0.5 mL/min and 40 °C. A Thermo Scientific Velos Pro ion trap with HESI-II probe operated in positive full MS mode.

Main Results and Discussion

Retention time precision across all systems and gradients was below 85 ms (n=199), with the steepest 2 min gradient showing the highest signal-to-noise. Reducing mixer volume to 35 µL maintained baseline stability and peak shape. High-pressure gradient systems achieved 199 samples/day for a 2 min method compared to 144 samples/day on the quaternary system. Using a 1.5 µm column and ramping flow from 1 to 1.25 mL/min increased backpressure to ~1300 bar and enabled 275 samples/day. An ultrafast 1.85 min gradient allowed 567 samples/day while preserving resolution and sub-second peak widths.

Benefits and Practical Applications

High-pressure systems with low gradient delay volumes maximize throughput and sensitivity for rapid screening workflows. Quaternary low-pressure gradient configurations offer flexibility for extended gradients and multiple solvent conditions, supporting diverse analytical requirements in regulated environments.

Future Trends and Opportunities

Advances in UHPLC hardware to reduce system dead volume and increase pressure stability will further enhance throughput. Integration with high-resolution MS and automated sample handling will expand applications in metabolomics, clinical diagnostics and real-time monitoring. Development of novel stationary phases and microfluidic mixers will push performance boundaries.

Conclusion

The three Vanquish systems exhibit overlapping performance, allowing selection based on gradient length, solvent flexibility and throughput demands. For ultrafast analysis and maximum sample turnover, high-pressure binary systems are recommended. For complex separations requiring multiple solvents, quaternary systems provide greater flexibility.

Used Instrumentation

• Thermo Scientific Vanquish Horizon UHPLC system
• Thermo Scientific Vanquish Flex Binary UHPLC system
• Thermo Scientific Vanquish Flex Quaternary UHPLC system
• Thermo Scientific Hypersil GOLD C18 column
• Thermo Scientific Accucore Vanquish C18+ column
• Thermo Scientific Velos Pro ion trap mass spectrometer with HESI-II probe